Mechanical loading of bone initiates an anti-catabolic and anabolic cellular response that promotes formation of a structurally competent skeleton. The work proposed in this competitive renewal will advance our study of the loaded response of the skeleton by examining a novel temporal sequence of gene regulation and deciphering whether orchestration of this anabolic process arises through a single initiating signal cascade. Our data reveal that mechanical strain regulates an early cluster consisting of canonical Wnt responders followed by a late cluster of anabolic genes, represented by Runx2, osterix (Osx) and eNOS. This pattern of strain response is mirrored by gene response to shear force suggesting that there is a prototypical biomechanical response. A common signaling pathway involving HRas/ERK1/2 is hypothesized to regulate those genes comprising the clustered response. This will be studied in SA1, comparing these candidate responses after strain and oscillatory shear. Our data further suggests a temporal pattern to the loading response: the canonical [unreadable]-catenin target response is vigorous at 4 h but returns to basal levels by 18 h while alterations in Runx2 and osterix are not measurable until 18 h after application of loading. Caveolin-1, a structural molecule in the lipid raft, regulates [unreadable]-catenin activity by limiting [unreadable]-catenin accessibility to signals that induce its nuclear translocation. Silencing caveolin-1 in osteoblasts accelerates load induced increase in Runx2 and Osx to within 4 hours of applying strain, an effect we propose occurs through enhancement of [unreadable]-catenin signaling. This suggests that [unreadable]-catenin may be important for later mechanical effects;causal relationships between early ([unreadable]-catenin targets) and late (requiring HRas/ERK1/2 activation) cell responses to mechanical stimulation are the subject of SA 2. In this aim we also track gene and cellular targets in bone after in vivo loading of both wild-type and caveolin-1 null mice to verify that these responses in the skeleton. Finally, SA3 will compare the global gene response between strain and shear in a temporal microarray to elucidate differential mechanical signals between the two forces, both in control cells, and in those where the putative early response (via [unreadable]-catenin) is altered. This will allow us to identify new signaling targets and verify those critical to the loaded response. The work proposed will utilize strain and oscillatory shear force applied to primary murine stromal cells and an osteoblast cell line in vitro, as well as in vivo loading of mice. Necessary cellular and molecular tools, and a caveolin-1 null mouse are in hand. In summary, our laboratory is in a strong position to bring novel insights into understanding the mechanisms by which loading generates an anti-catabolic and pro-anabolic response in bone cells. PUBLIC HEALTH RELEVANCE: The role of exercise to generate a functionally sufficient skeleton involves control of the differentiation of mesenchymal stem cells along the osteoblast lineage. The signaling cascades initiated by mechanical stimulation of bone cells confer a cellular phenotype that is both anti- catabolic and pro-anabolic. Work proposed here seeks to understand the loading induced signals and responses that result in this phenotype, thereby bringing novel insights into the mechanisms by which the loaded response is generated.